ABSTRACT G protein-coupled receptor (GPCR) signaling plays a critical role in the regulation of cardiac function under both resting and stressed conditions, and changes in GPCRs represent key characteristics of the failing heart. Signaling through GPCRs is tightly controlled by the actions of GPCR kinases (GRKs) and these enzymes have been implicated and proven to be mechanistically important in the pathogenesis of heart failure (HF). One GRK in particular, GRK2, which is up-regulated in ischemic and failing human myocardium, has been proven to be pathogenic in the heart. This appears, in part, due to the role of this enzyme in GPCR dysfunction, primarily ?- adrenergic receptor (?AR)-mediated contractility. Indeed, GRK2 inhibition or lowering of expression has reversed ?AR down-regulation in several HF models, restoring inotropic reserve. Thus, innovative ways to limit GRK2 activity in the failing heart appear to be translationally significant as a novel therapeutic approach to HF. GRK2's up-regulation at the transcriptional level has never been uncovered and we have new evidence that a microRNA (miR) program may be involved and offer insight into limiting GRK2 hyper-activity in the ischemic and injured heart. Importantly, GRK2 has an ever-expanding interactome demonstrating that increased GRK2 expression in the failing heart has pathological mechanisms that are non-GPCR dependent. These include our finding where GRK2 is a negative regulator of insulin signaling in the heart and more recently, we have found that GRK2 is a pro-death kinase when it is localized to mitochondria after ischemic injury/stress. Mitochondrial GRK2 also has non-canonical effects on myocyte metabolism. We have found that the pro-death effects of GRK2 are dependent on serine residue 670 (S670), which becomes phosphorylated by MAP kinases after ischemic stress inducing mitochondrial accumulation of GRK2. Therefore, limiting GRK2 accumulating in mitochondria after stress may be therapeutic and this will be tested herein. Since there is a pool of GRK2 in mitochondria at all times, it will also be important and significant to identify binding partners and potential substrates of this kinase mediated these novel effects on mitochondrial function and metabolism. Specifically, the Central Hypothesis of this competitive renewal is that GRK2 plays a critical role in the pathogenesis of cardiac contractile and metabolic dysfunction and HF via mechanisms beyond GPCR and ?AR desensitization. These mechanisms include the novel action of this enzyme within mitochondria. Further, elucidation of processes involved in the transcriptional control of GRK2 expression in the normal and injured heart will provide novel ways to limit its up-regulation, which may translate to significant new HF therapies. Specific Aims are: [1] To elucidate the role and mechanistic involvement of S670 phosphorylation of GRK2 in mitochondrial function and HF development after ischemic injury using a novel GRK2-S670A knock-in mouse model where all endogenous GRK2 cannot be regulated by MAP kinase; [2] To determine the role of GRK2 in cardiac metabolism and oxidative stress after ischemic injury in vivo, including elucidation of non-canonical binding partners and targets of GRK2 in cardiomyocyte mitochondria; [3] To determine novel miR-mediated regulation of GRK2 in the heart after ischemic stress and to determine its translational significance as a HF therapy.